jeremy j. johnson nih public access imtiaz a. siddiqui ...bacterial infections [7, 8]. in addition,...

Enhancing the bioavailability of resveratrol by combining it withpiperine

Jeremy J. Johnson1, Minakshi Nihal2, Imtiaz A. Siddiqui2, Cameron O. Scarlett3, Howard H.Bailey4,5, Hasan Mukhtar2,5, and Nihal Ahmad2,5

1Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago,Chicago, IL, USA2Department of Dermatology, School of Medicine and Public Health, University of Wisconsin-Madison, WI, USA3Analytical Instrumentation Center, School of Pharmacy, University of Wisconsin-Madison,Madison WI, USA4Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA5University of Wisconsin Carbone Cancer Center, Madison, WI, USA

AbstractScope—Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin shown to possess a multitude ofhealth-promoting properties in pre-clinical studies. However, the poor in vivo bioavailability ofresveratrol due to its rapid metabolism is being considered as a major obstacle in translating itseffects in humans. In this study, we examined the hypothesis that piperine will enhance thepharmacokinetic parameters of resveratrol via inhibiting its glucuronidation, thereby slowing itselimination.

Methods and results—Employing a standardized LC/MS assay, we determined the effect ofpiperine co-administration with resveratrol on serum levels resveratrol and resveratrol-3-O-β-D-glucuronide in C57BL mice. Mice were administered resveratrol (100 mg/kg; oral gavage) orresveratrol (100 mg/kg; oral gavage) + piperine (10 mg/kg; oral gavage), and the serum levels ofresveratrol and resveratrol-3-O-β-D-glucuronide were analyzed at different times. We found thatthe degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serumconcentration (Cmax) was increased to 1544% with the addition of piperine.

Conclusion—Our study demonstrated that piperine significantly improves the in vivobioavailability of resveratrol. However, further detailed research is needed to study the mechanismof improved bioavailability of resveratrol via its combination with piperine as well as its effect onresveratrol metabolism.

KeywordsAbsorption; Bioavailability; Pharmacokinetics; Piperine; Resveratrol

© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Correspondence: Professor Nihal Ahmad, Department of Dermatology, University of Wisconsin, 423 Medical Sciences Center, 1300University Avenue, Madison, WI 53706, USA, [email protected], Fax: +1-608-263-5223.

The authors have declared no conflict of interest.

NIH Public AccessAuthor ManuscriptMol Nutr Food Res. Author manuscript; available in PMC 2012 March 06.

Published in final edited form as:Mol Nutr Food Res. 2011 August ; 55(8): 1169–1176. doi:10.1002/mnfr.201100117.

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1 IntroductionResveratrol (3,5,4′-trihydroxystilbene; Fig. 1) is a naturally occurring phytoalexin(chemicals produced naturally by plants when under attack by pathogens) found in grapes,red wine, berries, peanuts and many other plants and has been shown to possess a multitudeof health-promoting properties [1–5]. The phenomenon of “French Paradox”, theobservation that French people suffer a relatively low incidence of coronary heart disease(despite having a diet relatively rich in saturated fats), has been attributed to theconsumption of red wine that contains resveratrol [6]. Historically, the medicinal use ofresveratrol containing herbs has been known for a long time. For example, the root of thePolygonum cuspidatum that contains resveratrol has been used in traditional Japanese,Chinese, and Korean medicine to prevent or remedy dermatitis, favus, hyperlipidemia, andbacterial infections [7, 8]. In addition, dietary supplements containing resveratrol often useP. cuspidatum standardized to resveratrol. These dietary supplements are being widelymarketed and used for a variety of health-related conditions. Thus, resveratrol is probablythe most common phytoalexin that has been used in traditional medicine as well as beinginvestigated for its possible use in modern health care.

Naturally occurring resveratrol is found in cis- and trans-chemical configurations; however,trans-configuration is the major configuration and also represents the most thoroughlystudied chemical form. In mice, resveratrol has been shown to be well tolerated at doses ashigh as 5000 mg/kg/day, for 28 consecutive days; however, high doses were associated withmortality due to the impaction of resveratrol in the gastrointestinal tract [9]. In healthyhuman volunteers, resveratrol has been administered at doses as high as 5 g as a single doseor at 5 g daily for up to 28 days in two different studies [10, 11]. Although peak serumresveratrol concentrations from a single 5-g dose is below the resveratrol concentrationsused and found effective in in vitro studies, this dose was shown to be well tolerated with noserious adverse events. In healthy human volunteers, 5 g of resveratrol with a meal achieveda peak plasma concentration of 539 ng/mL occurring 1.5 h post-dosing [10]. Interestingly,peak levels of two monoglucuronides and resveratrol-3-sulfate were three- to eightfoldhigher than resveratrol. Pharmacokinetic studies in animals and humans have identified twoattributes of resveratrol. First, the majority of trans-resveratrol metabolites were found to bederived by glucuronidation or sulfation, with at least seven different identified forms [12].Second, the metabolism of resveratrol has been shown to be very rapid with the majority ofbiotransformation occurring within the first hour following oral administration. Resveratrolmetabolites have very short half-life and are subjected to rapid urinary elimination. A fewclinical studies have suggested that the time to maximum serum concentration (Tmax) ofresveratrol could be impacted and delayed by certain types of foods [13, 14]. However,combining resveratrol with these foods did not significantly increase the absorption ofresveratrol. The rapid metabolism and poor pharmacokinetic parameters of resveratrol hasrepresented to be major obstacles in translating its observed therapeutic effect in pre-clinicalstudies to controlled clinical settings in humans [7].

Some studies have shown that piperine, an alkaloid derived from black pepper (Piper spp.),inhibits glucuronidation [15–17]. In this study, we examined the hypothesis that piperinewill enhance the in vivo bioavailability of resveratrol via inhibiting its glucuronidation,thereby slowing its metabolism.

2 Materials and methods2.1 Chemicals and reagents

Trans-Resveratrol (purity >95%) and piperine (purity >96.6%) were provided by Sabinsa(East Windsor, NJ, USA). Resveratrol-3-O-β-D-glucuronide (purity 98%) was obtained from

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Toronto Research Chemicals (North York, ON, Canada). Naproxen (purity >98.5%)meeting United States Pharmacopeia (USP) testing specifications was used as an internalstandard and purchased from Sigma (St. Louis, MO, USA). Chemical structures ofresveratrol, resveratrol-3-O-β-D-glucuronide, piperine, and naproxen is shown in Fig. 1.Liquid chromatography-mass spectrometry (LC-MS) grade solvents (water; CH3CN, ACN;CH3OH, methanol; (CH3)2CHOH, isopropanol; acetic acid, CH3COOH; ammoniumhydroxide, NH4OH; and ammonium bicarbonate, NH4HCO3) were obtained from FisherScientific (Pittsburgh, PA, USA). Blank plasma for preparation of the calibration curve andmethod development testing was obtained from the University of Wisconsin Hospital andClinics.

2.2 Sample preparationStandards were initially dissolved at >1 mg/mL in 100% ethanol. Because of the photolabilenature of resveratrol and resveratrol glucuronide, all samples were protected from light asmuch as possible during preparation and processing. For preparation of calibration curve,the internal standard and the standards were further diluted in 65% methanol before beingadded to samples. Seven point calibration curves (10, 33.3, 100.0, 333, 1000, 3333, and 10000 ng/mL resveratrol, 33.3, 100, 333, 1000, 3333, 10 000, and 33 333 ng/mL forresveratrol glucuronide) were prepared and processed in parallel with serum samples.

2.3 Instrumentation and LC/MS conditionsSamples were analyzed by LC separation on a BEH-C18 column (Waters, Milford, MA,USA) with water/1 mM ammonium bicarbonate pH 7.5/2% isopropanol as solvent A andACN/2% isopropanol as solvent B. Sample (10 µL) was injected onto the columnmaintained at 35°C that had been equilibrated at 5% B. After a 0.5-min hold at 5% Bcompounds were eluted with a linear gradient to 55% B in 6.5 min. The column was thenwashed by ramping to 95% B in 0.5 min, holding at 95% B for 1.5 min, and then the columnwas re-equilibrated at 5% B for 1.5 min before the next injection. Compounds eluting fromthe column were analyzed in negative ion mode with an Amazon ion-trap mass spectrometer(Bruker Daltonics, Billerica, MA, USA) following post-column addition of ammoniumhydroxide to 0.1%. Between samples, a blank injection of 35% ACN was included to ensurethat sample carry-over did not affect results. A typical elution profile is shown in the partialchromatogram in Fig. 2. Peaks were ~0.1 min wide (6 s), reflecting the high-resolution LCprovided by the BEH column.

2.4 Animals and treatmentsForty-eight C57BL/6J mice (10–12 wk old) were purchased from the National CancerInstitute at Frederick (Frederick, MD, USA). The mice were housed in plastic cages understandard conditions and received a standard chow and water ad libitum. The mice weremaintained on a 12 h/12 h light/dark cycle. Twenty-four mice (non-fasting) received a singledose of resveratrol (100 mg/kg via oral gavage; in 100 µL DMSO) while the other 24 mice(non-fasting) received combination resveratrol (100 mg/kg via oral gavage; in 50 µLDMSO) plus piperine (10 mg/kg via oral gavage; in 50 µL DMSO). All mice were weighedprior to experiment to ensure for accurate dosing of study agent(s). All animal experimentswere performed according to the policies and guidelines of the Institutional Animal Care andUse Committee (IACUC) of the University of Wisconsin.

2.5 Serum preparationAfter treatment with the test agents, at 0.25, 0.5, 1, 2, 4, 6, 12, and 24 h, blood samples werecollected from the mandibular vein following the administration of isoflurane as anesthetic.Three mice per time point per treatment group were used for obtaining blood. Blood samples

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were stored at room temperature for 30 min and were centrifuged at 4000 × g at 4°C for 15min for isolating serum [18]. Serum samples were transferred to 1.5-mL tubes and stored at−80°C until LC/MS analysis. Serum samples and calibrants (25 µL) were deproteinized bythe addition of 1.75 µL of glacial acetic acid and 30 µL of methanol followed by a 10-minvortex then incubation >1 h at −20°C. The mixture was subjected to centrifugation at 21 000× g and the supernatant (~30 µL) was diluted with 150 µL water and subjected to solid-phaseextraction (SPE) enrichment on an Oasis hydrophilic/lipophilic balanced (HLB) matrix(Waters) in a 96-well plate format. Briefly, each well of the plate was conditioned with 1mL of methanol, then 0.5 mL of 2 M acetic acid in water. Following conditioning, thesample was loaded. The plate was then washed with 0.5 mL of 2 M acetic acid followed by0.5 mL of 35% methanol/2 M acetic acid. The analytes were recovered by eluting with 0.5mL methanol with 1 M acetic acid. The samples were dried under nitrogen and suspended in30 µL of 35% ACN/65% water by vortexing 10 min.

2.6 LC/MS data analysisData were analyzed with the QuantAnalysis software (Bruker Daltonics, Billerica, MA,USA). Extracted ion chromatograms for the analytes and internal standard were created.Quadratic modeling of the analyte area under the curve relative to the internal standard areawas used to determine concentrations of resveratrol and resveratrol-3-O-β-D-glucuronide inanimal samples (Fig. 3). Quadratic curve fitting of the area under the curve for extracted ionchromatograms of the resveratrol and resveratrol-3-O-β-D-glucuronide relative to thenaproxen internal standard was used to determine analyte levels in samples. Analyte peakstypically had >30 data points/peak. Limits of detection were defined by analyte signals witha signal-to-noise ratio of 3. The limit of quantitation was defined as an analyte signal with asignal-to-noise ratio of 10. The percent recovery from plasma samples was calculated to be67% for resveratrol-3-O-β-D-glucuronide and 78% for resveratrol.

2.7 Pharmacokinetic parametersMean plasma concentrations of resveratrol and resveratrol-3-O-β-D-glucuronide were plottedversus time. The pharmacokinetic parameters were determined by non-compartmentalmethods. Area under the concentration-curve AUC0−inf was estimated from the time ofdosing and extrapolated to infinity. Analysis was performed using PKSolver 2.0 [19].

3 Results3.1 Method development and validation

As a first step, we first developed and standardized the LC/MS assay to analyze resveratroland resveratrol-3-O-β-D-glucuronide in plasma. For this purpose, human plasma sampleswere used. A typical chromatogram, containing naproxen spiked as an internal standard, isshown in Fig. 2. The retention times of resveratrol-3-O-β-D-glucuronide, naproxen, andresveratrol were 2.53, 3.73, and 4.14 min, respectively (Fig. 2).

We first analyzed five samples to test the small volume preparation method becauseprevious resveratrol sample analysis typically utilized 500 µL of starting material that wasnot feasible for animal studies in mice. We were able to adapt the SPE method for a smallstarting volume (25 µL). Calibration curves of resveratrol and resveratrol-3-O-β-D-glucuronide were constructed by plotting the relative peak area ratio (analyte/internalstandard) against the concentration of resveratrol or resveratrol-3-O-β-D-glucuronide. Thecalibration curve for resveratrol had an R value of 0.999793 (Fig. 3). In our assay condition,the limit of detection for resveratrol was found to be 21 ng/mL and the limit of quantitationwas 63 ng/mL. The calibration curve for resveratrol glucuronide had an R value of 0.999994(excluding the 3300 ng/mL calibration point). The limit of detection for resveratrol-3-O-β-D-

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glucuronide was 10 ng/mL and the limit of quantitation was 30 ng/mL. Thus, at the 24-htime point, resveratrol and resveratrol-3-O-β-D-glucuronide were below the limit ofquantification. Two samples analyzing resveratrol in the combination resveratrol andpiperine group were above the highest calibration point. The inter-day variation was 12.7%for resveratrol-3-O-β-D-glucuronide and 15.6% for resveratrol. The intra-day (i.e.repeatability) variation was 3.3% for the resveratrol-3-O-β-D-glucuronide and 1.1% forresveratrol.

3.2 Pharmacokinetics of resveratrolA method was developed and used successfully to support the pharmacokinetic study ofresveratrol in mice after a single oral dose of resveratrol or combination of resveratrol andpiperine. The mean serum concentrations of resveratrol and resveratrol-3-O-β-D-glucuronidefollowing resveratrol or resveratrol and piperine combination is shown in Fig. 4 and Tables1 and 2. Table 3 summarizes the main pharmacokinetic parameters of resveratrol andresveratrol-3-O-β-D-glucuronide in mice following administration of resveratrol orcombination of resveratrol and piperine and was calculated by non-compartmental analysis.Mice administered 100 mg/kg of resveratrol achieved a maximum serum concentration of2277 ng/µL at 0.25 h. The resveratrol metabolite resveratrol-3-O-β-D-glucuronide wascalculated to achieve a maximum serum concentration of 17 544 ng/µL at 0.25 h. The t1/2for resveratrol and resveratrol-3-O-β-D-glucuronide were 5.40 and 6.46 h, respectively. Thesecond cohort of mice, which received a combination of resveratrol (100 mg/kg) andpiperine (10 mg/kg), achieved a maximum serum concentration of resveratrol of 35 169 ng/L at 0.25 h. The t1/2 was calculated using the equation t1/2 = 0.693/Ke (i.e. Ke = eliminationrate constant). The AUC0–inf of resveratrol and resveratrol-3-O-β-D-glucuronide in cohortreceiving resveratrol only were 5046 and 89 794 ng/mL h, respectively. The AUC0–inf ofresveratrol and resveratrol-3-O-β-D-glucuronide in the cohort receiving combination ofresveratrol and piperine were 11 584 and 73 446 ng/mL h, respectively. Thus, our datasuggested that the degree of exposure to resveratrol was enhanced to 229% with the additionof piperine and the degree of exposure to the principal metabolite resveratrol-3-O-β-D-glucuronide was decreased to 81%. The Cmax of resveratrol was increased by 1544% withcombination resveratrol and piperine. The Tmax of resveratrol-3-O-β-D-glucuronide was 0.5h with combination of resveratrol and piperine while the resveratrol only cohort had a Tmaxof 0.25 h.

4 DiscussionThe major finding of this study is that piperine significantly enhances the serumbioavailability of resveratrol in mice. Because the rapid metabolism seems to be a limitingfactor in translating resveratrol’s promising health-promoting effects in humans, in thisstudy we investigated our recently propagated strategy to enhance the pharmacokineticparameters of resveratrol by partially inhibiting its metabolism via combining it withpiperine [7].

Studies in healthy human volunteers have shown that resveratrol is rapidly absorbed butquickly metabolized, usually within 30–60 min following oral or intravenous administrationin humans [10, 20]. Similarly, in mice, oral resveratrol (240 mg/kg body weight) has beenreported to achieve a Cmax of ~32 µM (n = 3) in 10 min and a AUC of 863 nm/mL min [21].In our study, we observed oral resveratrol (100 mg/kg body weight) to achieve a Cmax of~12 µM (n = 3) at 15 min and an AUC of 368 nm/mL min. When taking into account thedifference in dose (240 mg/kg versus 100 mg/kg) and time point (10 versus 15 min), ourdata appear to be consistent with the study by Sale et al., described above [21].

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A few studies have shown that piperine inhibits glucuronidation of certain polyphenolicantioxidants, viz. curcumin and epigallocatechin-3-gallate [15–17, 22–24]. Resveratrol hasbeen shown to be extensively metabolized through glucuronidation and sulfation. In thisstudy, we found that piperine appreciably modulates the pharmacokinetics of resveratrol byincreasing the Cmax and the AUC. We have found that piperine increase Cmax of resveratrolby 1544%, Cmax of resveratrol-O-β-D-glucuronide by 184%, and an increase in the Tmaxfrom 0.25 h to 0.5 h. The delay in the glucuronidation of resveratrol to resveratrol-O-β-glucuronide is suggestive that piperine inhibited UGT1A1, which has been shown to beresponsible for the formation of 3-O-β-glucuronide [25]. In future studies, we plan toevaluate the effect on additional metabolites to determine the impact of piperine on otherUGT enzymes such as UGT1A9, which is responsible for the formation of resveratrol-4-O-β-glucuronide. The observed increase in the absorption of resveratrol (i.e. 1544%) by theaddition of piperine further suggests toward the possibility of inhibition of glucuronidationin the GI tract. To further support our hypothesis of piperine having a local GI effect, we didnot observe an increase in the half-life of resveratrol suggesting the systemic absorption ofpiperine may not be a critical attribute. It is possible that piperine also modulates theintestinal membrane dynamics by influencing brush border membrane fluidity [26].Interestingly, in our study, we observed the presence of a second peak in serum with theresveratrol metabolite, which may be explained by enterohepatic recirculation of resveratrol,which has been observed in rats and humans [20, 27]. Furthermore, piperine is known todecrease gastrointestinal emptying and increase transit time, which could be responsible forits observed response [28]. Piperine has been shown to be metabolized by oxidation of thepiperidine ring to form a 3,4-dihydroxyphenyl motif, which may allow for glucuronidationand elimination. Interestingly, resveratrol has been shown to inhibit phase I and phase IIenzymes and may actually alter the pharmacokinetics of piperine [29, 30]; however, furtherstudies are needed to support this possibility.

In conclusion, our study represents the first evaluation of piperine modulating thepharmacokinetic parameters of resveratrol. Our data indicate that piperine increases theCmax and degree of exposure (i.e. AUC) to resveratrol, which appears to be through theinhibition of glucuronidation of resveratrol by piperine. While mechanistic details of thiseffect remain to be elucidated, our study could have direct application to human studiesevaluating the pharmacokinetic profile of resveratrol. For instance, clinical studies have useddoses as high as 5 g daily, which is the equivalent of ten capsules (500 mg each). Thisrepresents a significant shortcoming in the long-term translational development ofresveratrol as described above. One study in particular has shown that 1 g of resveratrol,which is the equivalent of two capsules, was administered daily for 28 days and was welltolerated [29]. All documented adverse events were grade 1 or 2 Common Toxicity Criteria,with many being very mild and transient. Given our results, the strategy of piperine co-administration may allow for a significant decrease in the dose of resveratrol (and capsules)in clinical settings. As a result, we are currently in the process of enrolling subjects into aphase I clinical trial in healthy volunteers to study the effect of piperine on thebioavailability of resveratrol in a clinical setting. Finally, piperine has been shown to be welltolerated in animals and humans; however, it is possible that through the inhibition ofglucuronidation, there could the potential to increase the degree of exposure (i.e. AUC) topharmaceuticals metabolized by glucuronidation. To minimize the systemic effects ofpiperine, our future work is focused on optimizing the dose of piperine to ensure localgastrointestinal effects as a strategy to prevent adverse drug interactions.

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AcknowledgmentsThe authors thank Sabinsa, Inc. and Dr. Muhammad Majeed for generously providing resveratrol and piperine forthese studies. This work was partially supported by funding from the National Institutes of Health (1R21CA149560, R01CA114060, and 1R01AR059130).

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Figure 1.Structures of resveratrol, resveratrol-3-O-β-D-glucuronide, piperine, and internal standardnaproxen.

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Figure 2.Elution profiles in a typical chromatogram for the three analytes. Extracted ionchromatograms for resveratrol 3-O-β-D-glucuronide (403.1), a naproxen fragment (170.1 m/z), and resveratrol (227.1 m/z) are shown for a calibration point.

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Figure 3.Calibration curves for resveratrol (top) and resveratrol-3-O-β-D-glucuronide (bottom). Blankplasma samples were spiked with resveratrol, resveratrol-3-O-β-D-glucuronide, andnaproxen. The samples were purified using Oasis HLB SPE and analyzed by LC/MS.Quadratic curve fitting (black line) of the area under the curve for extracted ionchromatograms of the resveratrol and resveratrol-glucuronide relative to the naproxeninternal standard was used to determine analyte levels in animal samples. Internal standardareas for calibrants fell within three standard deviations of the average (grey points andlines).

Johnson et al.


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Figure 4.Mean concentration-time profile for resveratrol (A) and resveratrol-3-O-β-D-glucuronide(B). Mice were administered resveratrol (100 mg/kg) or combination of resveratrol (100 mg/kg) and piperine (10 mg/kg). Error bars represent SE. For a detail of exact value, please seeTables 1 and 2.

Johnson et al.


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Johnson et al.

Table 1

Average serum levels of resveratrol in C57Bl/6 mice following administration of resveratrol (100 mg/kg) orcombination resveratrol (100 mg/kg) plus piperine (10 mg/kg)

Hours Serum resveratrol (ng/mL)

Resveratrol(100 mg/kg)

Resveratrol and piperine(100 mg/kg and 10 mg/kg)

Mean SE Mean SE

0.25 2277.3 776.4 35 169.1 19 316.9

0.5 374.9 50.8 910.9 126.3

1 801.74 312.7 96.4 7.0

2 353.1 68.8 181.1 33.0

4 321.2 63.1 117.2 5.6

6 243.3 23.8 144.8 22.9

12 112.8 14.1 83.9 5.4

24 41.7 12.1 41.4 12.0


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Johnson et al.

Table 2

Average serum levels of resveratrol-O-β-D-glucuronide in C57Bl/6 mice following administration ofresveratrol (100 mg/kg) or combination resveratrol (100 mg/kg) plus piperine (10 mg/kg)

Hours Serum resveratrol-O-β-D-glucuronide (ng/mL)

Resveratrol(100 mg/kg)

Resveratrol and piperine(100 mg/kg and 10 mg/kg)

Mean SE Mean SE

0.25 17544.3 1436.1 23 324.9 4387.8

0.5 10 639.2 181.1 32 400.5 7093.9

1 2705.7 320.3 2175.2 348.6

2 6914.4 1158.9 2997.6 787.2

4 7256.9 585.2 4798.5 1045.6

6 4806.0 692.5 3377.1 184.2

12 2803.5 1415.3 2887.7 654.5

24 656.2 339.6 23.4 2.2


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Table 3

Pharmacokinetic parameters of resveratrol and resveratrol-3-O-β-D-glucuronide following administration ofresveratrol (100 mg/kg) or combination resveratrol (100 mg/kg) plus piperine (10 mg/kg)

Pharmacokinetic parametersa)

Cmax (ng/mL) Tmax (h) Half-life (h) AUC0–inf (ng/mLh)

Resveratrol cohort

Resveratrol 2277.27 0.25 5.40 5046.3

Resveratrol-3-O-β-D-glucuronide 17 544.26 0.25 6.46 89 793.70

Resveratrol and piperine cohort

Resveratrol 35 169.07 0.25 4.84 11 583.70

Resveratrol-3-O-β-D-glucuronide 32 400.54 0.50 3.03 73 446.60

a)Data were calculated using non-compartmental analysis.


jeremy j. johnson nih public access imtiaz a. siddiqui ...bacterial infections [7, 8]. in addition,...

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